This application relates to a novel form of separation matrix for the analysis of biopolymers, particularly nucleic acid polymers.
The use of separation matrices for the analysis of biopolymers is well established. For example, agarose gels, polyacrylamide gel and other types of gel matrices are used routinely to separate proteins, polypeptides and polynucieotides into subclasses based upon properties such as size, weight and molecular charge. Analysis of the separated subclasses is used to identify and characterize proteins, to detect and characterize mutant forms of proteins, and to detect and characterize polynucleotides. For example, analysis of separated polynucieotide fragments is a basic part of the process for most determinations of nucleic acid sequence.
Although the gel matrices which have been used for these separations to date are effective and can produce useful analytical results, they are not without their deficiencies. These deficiencies include (1) a randomness of structure, and (2) a requirement for continuous hydration after formation. Both of these deficiencies can lead to unpredictable variations in the results obtained between one gel and another, whether as a result of intrinsic variations in the gels, or as a result of changes resulting from differing storage conditions. In addition, the micro-inhomogeneity resulting from randomness of the gel structure reduces the actual resolving power of the gels. A further deficiency of known gels used in separations of biopolymers arises from the nature of the gel materials themselves. The materials used may be subject to breakdown by high electric fields, thus limiting the field strength that could otherwise be employed to obtain more rapid separation. In addition, the gel, or materials used in forming the gel such as accelerators, may interfere with optical detection of the separated biopolymers.
Volkmuth et al, Nature 358: 600-602 (1992) have proposed the use of a SiO.sub.2 obstacle course fabricated using optical microlithography for the analysis of large DNA molecules having a length on the order of 100 kilobases. The obstacle course is made up of a regular array of posts having separations of 1 .mu.m between the posts. DNA loaded onto the array was separated by size by the application of an electric field, and detected using epifluorescence microscopy.
The obstacle course described by Volkmuth et al. is not well-suited for use in diagnostic applications, however, because the total length of the DNA fragments in most diagnostic DNA sequencing applications, diagnostic RFLP (restriction fragment length polymorphism) procedures, and the like is between about 20 and 300 to 400 nucleotides. As such, the looping of long strands of DNA observed by Volkmuth cannot be relied upon as a basis for separation. Furthermore, the fabrication process of Volkmuth et al. uses electron beam lithography to make each individual device, which would be prohibitively expensive for diagnostic applications.
International Patent Publication No. WO94/29707 discloses a microlithographic array for macromolecule and cell fractionation. The array is made using photolithography and then used for separation of macromolecules or cells migrating under the influence of an electric, magnetic, hydrodynamic or optical field.
It is an object of the present invention to provide an alternative from of separation matrix which overcomes these deficiencies of known gel matrices and which is useful in the analysis of smaller DNA fragments useful in diagnostic applications.
It is a further object of the present invention to provide a separation matrix for the separation and analysis of biopolymers which has a highly regular structure.
It is still a further object of the present invention to provide a separation matrix which can withstand very high electric field strengths.
It is still a further object of the present invention to provide a separation matrix which permits separated biopolymers to be optically detected with very high efficiency.
It is still a further object of the invention to provide a method for manufacturing the novel separation matrices of the invention.
It is still a further object of the present invention to provide a method for separating biopolymers, and in particular for separating oligonucleotide fragments for purposes of analyzing DNA sequences, using the novel separation matrices of the invention.